Anthrax toxin protective antigen protein (PA) binds to receptors on the surface of mammalian cells, is cleaved by cellular proteases, forms an oligomer, and transports two other toxin proteins, lethal factor (LF) or edema factor (EF) to the cytosol. EF is a potent calmodulin-dependent adenylyl cyclase that causes large increases in intracellular cAMP concentrations resulting in localized and systemic edema and activation of pathways downstream of protein kinase A (PKA) and Epac/Rap1-signaling. LF is a metalloprotease that cleaves and inactivates several mitogen-activated protein kinase kinases (MEKs). In rodents LF also cleaves and activates inflammasome sensor NLRP1. The inflammasomes are intracellular complexes that play a role in innate immune sensing for defense against pathogens. The cleavage of NLRP1 in macrophages and dendritic cells leads to caspase-1 activation, a rapid cell death termed pyroptosis, maturation and release of the pro-inflammatory cytokines IL-1 and IL-18 and recruitment of innate immune cells. Rat NLRP1 but not murine Nlrp1 is also activated by Toxoplasma gondii but the mechanism for its activation or differential responses between rodent strains is currently unknown. The inhibition of the MEK pathways and NLRP1 cleavage-mediated activation of the immune response have a wide range of consequences for the host and are believed to account for some of the actions of LT during disease. In the last year we have continued work on a new substrate for anthrax LF and verified cleavage of this substrate in different cell types. We identified mutant variants of this substrate that are resistant to cleavage. The cleavage was linked to shutdown of major signaling events in cells. Mouse studies show the cleavage of this novel substrate may be a primary event linked to lethality induced by LF in vivo. The discovery of a new substrate for LF and shutdown of multiple important signaling pathways is a major step forward in understanding anthrax pathogenesis and the mechanism by which this toxin disables many immune cell functions. We now have a better understanding of how B. anthracis kills the host. These findings also suggest new pathway targets for development of therapeutics. Collaborative studies during 2018 with UCSD continued investigation of the signaling mechanisms involved in EF's impact on cells. EF disrupts endocytic recycling by the GTPase Rab11 and prevents cadherin delivery to cellular junctions. We found that EF blocks Rab11 after GTP loading. The Epac pathway which is activated by EF-induced cAMP blocks fusion of recycling endosomes with membranes through actions of the small GTPase Rap1. A different small GTPase, Arf6, counteracts Rab 11 delivery of cargo proteins to junctions. In a mouse footpad edema model, we showed that pharmacological inhibition of the EPAC pathway, Arf6 or Rap1 activation significantly reduces toxin-induced edema, suggesting novel therapeutic targets for this toxin. These studies offer a new understand of how EF disrupts barrier function. In follow up collaborative studies during 2018, EF's impact on a new signaling pathway has been linked to the edema induced by this toxin in mice. Studies are underway to test pharmaceutical interventions in this novel pathway as a treatment against anthrax-induced edema. Preliminary findings suggest these pathways play a more significant role in control of toxin-induced junctional changes leading to edema than previously implicated pathways. These studies are actively underway. We continue long term studies using the Collaborative Cross recombinant inbred mouse collection to map susceptibility loci controlling sensitivity to anthrax LT in mice. This mouse collection is a unique resource - a large panel of inbred strains developed from eight unique parental founders which include three wild strains. The collection represents a wider genetic diversity than in any other inbred model, with segregating polymorphisms at every 100-200 bp. We are in the process of analyzing the genetic basis for unique LT-induced phenotypes present in this collection. In the last year we have mapped a locus in which absence of function is required for extreme sensitivity. New crosses are underway for mapping the additional loci controlling responses to LT. We also continued our studies on the role of inflammasome activation in both murine and rat resistance to T. gondii, as a parallel complement to our LT studies. In the past year we have found that IL-1 and IL-18 responses to inflammasome activation play very different roles in resistance to this parasite in mice and rats. The dissection of the cell types involved in inflammasome control of T. gondii is underway. In collaboration with colleagues at USDA, we have investigated the responses of NLRP1 proteins in the natural bovine hosts of anthrax. In preparation for these studies, we published a comprehensive review of what is known about all inflammasome responses in livestock and wildlife. Finally, LT is known to require cathepsin release and lysosomal membrane permeabilization for activation of the NLRP1 inflammasome. In a collaborative study, we found that a similar release of lysosomal cathepsin is required for activation of the NLRP3 inflammasome by Mycobacterium tuberculosis. Elevated levels of cathepsin B were found in lungs of mice and rabbit infected with this bacterium as well as in plasma from patients. The bacterium induces release of the cathepsin in an ESAT-6 dependent manner and the inhibitor CA074Me abrogates NLRP3 activation, caspase-1 activation and cytokine release in infected macrophages.